Cuini Mo (Summer 2008)

For this 2008 summer internship,
I have worked with Dr. Diego Loayza in his lab. We studied telomere
replication in human cells. Telomere is an important parameter in
cancer. They are made up of the sequence TTAGGG repeated thousands of
time. Telomeres end with a single stranded overhang. And to that
single stranded overhang, there is an enzyme called telomerase that
will synthesize the TTAGGG repeat sequences. Telomere is also made up
of specific proteins that bind to the repeat sequences TTAGGG and this
complex is called shelterin.

The goal of our lab was
to study C-stranded synthesis because it is required for telomere
maintenance and elongation, in addition to the necessary action of
telomerase. To achieve this goal, we started the analysis of known DNA
replication components at telomeres. We focused on FEN1 and POLA2
because they are known to be essential for C-strand synthesis in
yeast.

We asked whether FEN1 or
POLA2 were associated with specific shelterin components at telomeres.
To this end, we did a co-immunoprecipitation experiment. We detected
FEN1 in a complex with POT1 in HTC75 cells, in a MYC-FEN1
overexpressing cell line. A negative control included a
non-overexpressing cell line (vector only). This experiment shows that
FEN1 and POT1 are together in a complex in HTC75 cells.

We also performed
chromatin immunoprecipitations (ChIP). The result of this experiment
was that FEN1, TRF1, POT1 and possibly POLA2 were detected. This was
done by using the probe TTAGGG to detected telomeric sequences. An Alu
repeat probe was used as a negative control for internal chromosomal
sequences.

For future experiments,
we plan on studying the modalities of the FEN1-shelterin interaction,
and the significance of this interaction in telomere function.